A method of determining endotoxin using limulus amebocyte lysate (hereinafter abbreviated as lysate) is known. This method utilizes the coagulation of the lysate with a trace amount of endotoxin. The subsequent biochemical analysis reveals that the coagulation reaction is caused by a stepwise activation of several coagulation factors (Takanori Nakamura et al., Japanese Journal of Bacteriology, 38, 781-803 (1983)).
That is, as shown in FIG. 1, when endotoxin is added to the lysate, factor C (endotoxin-sensitive factor, molecular weight: 123,000) is activated to form an activated factor C. This activated factor C restrictedly hydrolyzes factor B (molecular weight 64,000), whereby the factor B is activated to form an activated factor B, which activates a proclotting enzyme (molecular weight: 54,000) to change into a clotting enzyme. The clotting enzyme restrictedly hydrolyzes specific sites Of Arg.sup.18 -Thr.sup.19 and Arg.sup.46 -Gly.sup.47 of coagulogen (coagulating protein, molecular weight: 19,723) to liberate a peptide C. The coagulogen is converted to coagulin, which causes coagulation (gelation). It is known that endotoxin can be quantitatively determined by the method of Iwanaga et al., (Haemostasis, 7, 183-188 (1978)) in which the lysate is used in combination with a synthetic peptide having an amino acid sequence corresponding to that of the hydrolyzed site of this coagulogen, i.e., the chromogenic substrate Boc-Leu-Gly-Arg-p-nitroanilide (pNA) or fluorogenic substrate Boc-Leu-Gly-Arg-4-methylcoumaryl-7-amide. This determination method utilizes a series of a reaction in which endotoxin triggers of a cascade mechanism for subsequently activating plural coagulation factors (which are all serine protease precursors) to finally form a coagulin gel. When (1.fwdarw.3)-.beta.-D-glucan is added to the lysate, factor G is activated to an activated factor G, which functions to convert the proclotting enzyme to the clotting enzyme as shown in FIG. 1. Then, as in the case of endotoxin, the clotting enzyme functions to convert coagulogen to a coagulin which forms a gel and hydrolyzes a synthetic substrate (Morita et al., FEBS Lett., 129, 318-321 (1981)).
The substances which react with factor G include (1.fwdarw.3)-.beta.-D-glucan, krestin, lentinan, and substances contained in the rinses from cellulosic hemodialyzer and in the blood contacted with the dialyzer. These substances are confirmed to exhibit no pyrogenesity by the rabbit pyrogenic test.
On the other hand, (1.fwdarw.3)-.beta.-D-glucan is known as a polysaccharide for constituting a mycotic cell wall. It is feasible to detect the presence of mycetes within the body by determining (1.fwdarw.3)-.beta.-D-glucan in blood, and therefore, a method for accurate and reproducible determination of (1.fwdarw.3)-.beta.-D-glucan without any interference of endotoxin is desired, particularly, in the field of clinical diagnosis.
Further, it is reported that seventeen anti-factor C monoclonal antibodies are prepared and these antibodies are examined for identification of their epitope regions and an effect for the activation of factor C (Yoshiki Miura et al., Biochemistry, 61, No. 9, 834 "Identification of monoclonal antibodies to lp-Ajo6 lipopolysaccharide-sensitive serine protease precursor (factor C) and application thereof to analysis of activation mechanism", Sep. 25, 1989, published by The Japanese Biochemical Society).
Further, a method of determining (1.fwdarw.3)-.beta.-D-glucan using factor G in the lysate has been reported (Obayashi et al., Clin. Chim. Acta. 149, 55-65 (1985)). This method comprises fractionating the lysate by gel filtration or affinity chromatography using a carrier having heparin or dextran sulfate fixed thereon to remove endotoxin-sensitive factor C and recombining only factor G and proclotting enzyme. Thus, the method requires extremely complicated operation.